Biology subjects

Travis, S. M.

Publications and source records attributed to Travis, S. M..

7 recordsLinked to original sources

bioRxiv2024

Mechanism of how the universal module XMAP215 γ-TuRC nucleates microtubules

It has become increasingly evident in recent years that nucleation of microtubules from a diverse set of MTOCs requires both the {gamma}-tubulin ring complex ({gamma}-TuRC) and the microtubule polymerase XMAP215. Despite their essentiality, little is known about how these nucleation factors interact and work together to generate microtubules. Using biochemical domain analysis of XMAP215 and structural approaches, we find that a sixth TOG domain in XMAP215 binds {gamma}-TuRC via {gamma}-tubulin as part of a broader interaction involving the C-terminal region. Moreover, TOG6 is required for XMAP215 to promote nucleation from {gamma}-TuRC to its full extent. Interestingly, we find that XMAP215 also depends strongly on TOG5 for microtubule lattice binding and nucleation. Accordingly, we report a cryo-EM structure of TOG5 bound to the microtubule lattice that reveals promotion of lateral interactions between tubulin dimers. Finally, we find that while XMAP215 constructs effects on nucleation are generally proportional to their effects on polymerization, formation of a direct complex with {gamma}-TuRC allows cooperative nucleation activity. Thus, we propose that XMAP215s C-terminal TOGs 5 and 6 play key roles in promoting nucleation by promoting formation of longitudinal and lateral bonds in {gamma}-TuRC templated nascent microtubules at cellular MTOCs.

biochemistry↗

bioRxiv2023

Conformational states of the microtubule nucleator, the γ-tubulin ring complex

Microtubules (MTs) perform essential functions in the cell, and it is critical that they are made at the correct cellular location and cell cycle stage. This nucleation process is catalyzed by the {gamma}-tubulin ring complex ({gamma}-TuRC), a cone-shaped protein complex composed of over 30 subunits. Despite recent insight into the structure of vertebrate {gamma}-TuRC, which shows that its diameter is wider than that of a MT, and that it exhibits little of the symmetry expected for an ideal MT template, the question of how {gamma}-TuRC achieves MT nucleation remains open. Here, we utilized single particle cryo-EM to identify two conformations of {gamma}-TuRC. The helix composed of 14 {gamma}-tubulins at the top of the {gamma}-TuRC cone undergoes substantial deformation, which is predominantly driven by bending of the hinge between the GRIP1 and GRIP2 domains of the {gamma}-tubulin complex proteins. However, surprisingly, this deformation does not remove the inherent asymmetry of {gamma}-TuRC. To further investigate the role of {gamma}-TuRC conformational change, we used cryo electron-tomography (cryo-ET) to obtain a 3D reconstruction of {gamma}-TuRC bound to a nucleated MT, providing insight into the post-nucleation state. Rigid-body fitting of our cryo-EM structures into this reconstruction suggests that the MT lattice is nucleated by spokes 2 through 14 of the {gamma}-tubulin helix, which entails spokes 13 and 14 becoming more structured than what is observed in apo {gamma}-TuRC. Together, our results allow us to propose a model for conformational changes in {gamma}-TuRC and how these may facilitate MT formation in a cell.

molecular biology↗

bioRxiv2023

Structure of a Membrane Tethering Complex Incorporating Multiple SNAREs

Most membrane fusion reactions in eukaryotic cells are mediated by membrane tethering complexes (MTCs) and SNARE proteins. MTCs are much larger than SNAREs and are thought to mediate the initial attachment of two membranes. Complementary SNAREs then form membrane-bridging complexes whose assembly draws the membranes together for fusion. Here, we present a cryo-EM structure of the simplest known MTC, the 255-kDa Dsl1 complex, bound to the two SNAREs that anchor it to the endoplasmic reticulum. N-terminal domains of the SNAREs form an integral part of the structure, stabilizing a Dsl1 complex configuration with remarkable and unexpected similarities to the 850-kDa exocyst MTC. The structure of the SNARE-anchored Dsl1 complex and its comparison with exocyst reveal what are likely to be common principles underlying MTC function. Our structure also implies that tethers and SNAREs can work together as a single integrated machine.

biochemistry↗

bioRxiv2022

RanGTP regulates the augmin complex

Spindles are composed of microtubules that must nucleate at the right place and time during mitosis. Spindle microtubule nucleation is regulated by the GTPase Ran, which, through importin-{beta}, releases a gradient of spindle assembly factors (SAFs) centered at chromosomes. Branching MT nucleation generates most spindle MTs and requires the augmin complex. In Xenopus laevis, Ran can control branching through the SAF TPX2, TPX2 is non-essential in other organisms. Thus, how Ran regulates branching MT nucleation in the absence of TPX2 is unknown. Here, we use in vitro pulldowns and TIRF microscopy to show that augmin is itself a SAF. Augmin directly interacts with both importins through two nuclear localization sequences on the Haus8 subunit, which overlap the MT binding site. Moreover, Ran controls localization of augmin to MTs in both Xenopus egg extract and in vitro. By uncovering that RanGTP directly regulates augmin, we demonstrate how Ran controls branching MT nucleation and, thereby, spindle assembly and cell division.

biochemistry↗

bioRxiv2022

Integrated Model of the Vertebrate Augmin Complex

Accurate segregation of chromosomes is required to maintain genome integrity during cell division. This feat is accomplished by the microtubule-based spindle. To build a spindle rapidly and with high fidelity, cells take advantage of branching microtubule nucleation, which exponentially amplifies microtubules during cell division. Branching microtubule nucleation relies on the hetero-octameric augmin complex, but understanding how augmin promotes branching has been hindered by a lack of structural information about the complex. Here, we report an integrated model of vertebrate augmin, combining cryo-electron microscopy, advanced protein structural prediction, and the visualization of fused bulky tags via negative stain electron microscopy. This strategy allowed us to identify the location and orientation of each subunit within the structure. Evolutionary analysis of augmins structure reveals that it is highly conserved across diverse eukaryotes, and that augmin contains a previously-unidentified microtubule binding site. Moreover, we identify homology with the kinetochore-localized NDC80 complex. This new model of the augmin complex provides insight towards the mechanism and evolution of branching microtubule nucleation.

molecular biology↗

bioRxiv2022

The conserved centrosomal motif, γTuNA, forms a dimer that directly activates microtubule nucleation by the γ-tubulin ring complex (γTuRC).

1.To establish the microtubule cytoskeleton, the cell must tightly regulate when and where microtubules are nucleated. This regulation involves controlling the initial nucleation template, the {gamma}-tubulin ring complex ({gamma}TuRC). Although {gamma}TuRC is present throughout the cytoplasm, its activity is restricted to specific sites including the centrosome and Golgi. The well-conserved {gamma}-tubulin nucleation activator ({gamma}TuNA) domain has been reported to increase the number of microtubules generated by {gamma}TuRCs. Here we utilize Xenopus egg extract and in vitro single molecule imaging assays to show that {gamma}TuNA activates microtubule nucleation in extract and directly activates {gamma}TuRC in vitro. Via mutation analysis, we find that {gamma}TuNA is an obligate dimer. Moreover, efficient dimerization as well as {gamma}TuNAs L70, F75, and L77 residues are required for binding to and activation of {gamma}TuRC. Finally, we find that {gamma}TuNAs activating effect opposes inhibitory regulation by stathmin. In sum, our study illuminates how {gamma}TuRC is controlled in space and time in order to build specific cytoskeletal structures.

biochemistry↗

bioRxiv2020

Structural basis for the binding of SNAREs to the multisubunit tethering complex Dsl1

Multisubunit tethering complexes (MTCs) are large (250 to >750 kDa), conserved macromolecular machines that are essential for SNARE-mediated membrane fusion in all eukaryotes. MTCs are thought to function as organizers of membrane trafficking, mediating the initial, long-range interaction between a vesicle and its target membrane and promoting the formation of membrane-bridging SNARE complexes. Previously, we reported the structure of the Dsl1 complex, the simplest known MTC, which is essential for COPI-mediated transport from the Golgi to the endoplasmic reticulum (ER). This structure suggested how the Dsl1 complex might function to tether a vesicle to its target membrane by binding at one end to the COPI coat and at the other end to ER SNAREs. Here, we use x-ray crystallography to investigate these Dsl1-SNARE interactions in greater detail. The Dsl1 complex comprises three subunits that together form a two-legged structure with a central hinge. Our results show that distal regions of each leg bind N-terminal Habc domains of the ER SNAREs Sec20 (a Qb-SNARE) and Use1 (a Qc-SNARE). The observed binding modes appear to anchor the Dsl1 complex to the ER target membrane while simultaneously ensuring that both SNAREs are in open conformations with their SNARE motifs available for assembly. The proximity of the two SNARE motifs, and therefore their ability to enter the same SNARE complex, depends on the relative orientation of the two Dsl1 legs.

cell biology↗